Gene probe assays which depend upon the binding of DNA probes with their complementary base pairs on a target molecule are among the most common assays used by molecular biologists. Such assays require a great deal of time to bind the probe to a target DNA, to remove any excess unhybridized probe and to analyze the hybridized probe.
The time required to perform these assays and the difficulties associated in performing these assays make such assays inefficient in searching for single nucleotide polymorphisms (SNPs). An SNP is a change (deletion, insertion or substitution) in any single nucleotide base in the region of the genome of interest. Because SNPs occur so frequently in the human genome (about once in every 500 bases), SNPs are useful markers in studying the human genome.
The detection of SNPs is typically performed using automated DNA sequencers. However such sequencers are generally not well utilized in determining a single base change within a 500 base sequence. As a result an SNP search using a sequencer is slow and expensive.
The recent development of Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has permitted small amounts (about 5 femtomoles) of DNA to be analyzed to extreme resolution (one dalton accuracy). The sensitivity and mass resolution of MALDI-TOF mass spectrometry fall off with increasing mass, so that the current upper practical limit for analysis of DNA is between about 50 to 100 nucleotide bases. Samples of 500 nucleotide bases have been analyzed with MALDI-TOF but with poor resolution and sensitivity.
The present invention relates to a method for quickly determining polynucleotide sequences, with low labor intensity and a low cost per SNP assay.